Purification and characterization of two beta-glucosidases from the thermophilic fungus Thermoascus aurantiacus

beta-Glucosidase from the fungus Thermoascus aurantiacus grown oil semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps - ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, beta-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80 degreesC. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl beta-D-glucopyranoside as substrate, K-m, values of 1.17 +/- 0.35 and 1.38 +/- 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

Distribution of I-125-labeled crotamine in mice tissues

Crotamine is a strong basic polypeptide from Crotalus durissus terrificus (Cdt) venom composed of 42 amino acid residues tightly bound by three disulfide bonds. It causes skeletal muscle spasms leading to spastic paralysis of hind limbs in mice. The objective of this paper was to study the distribution of crotamine injected intraperitoneally (ip) in mice. Crotamine was purified from Cdt venom by gel filtration, followed by ion exchange chromatography, using a fast-performance liquid chromatography (FPLC) system. Purified crotamine was irradiated at 2 kGy in order to detoxify. Both native and irradiated proteins were labeled with 125, using chloramine T method, and separated by get filtration. Male Swiss mice were injected ip with 0.1 mL (2 x 10(6) cpm/mouse) of I-125 native or irradiated crotamine. At various time intervals, the animals were killed by ether inhalation and blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine the radioactivity content. The highest levels of radioactivity were found in the kidneys and the liver, and the lowest in the brain. (c) 2006 Elsevier Ltd. All rights reserved.

Purification and properties of buffalo (Bubalus bubalis) erythrocyte hexokinase

Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A single protein band was detected by means of Western blotting using an antibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 +/- 2880 by gel filtration. Partial purification of erythrocyte hexokinase by a combination of several procedures, including affinity chromatography, which was previously applied successfully to the purifica tion of other mammalian type I hexokinases, produced a partially purified enzyme that showed several contami nants after SDS-polyacrylamide gel electrophoresis. The affinity of buffalo erythrocyte hexokinase for glucose (K-m = 0.012 +/- 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher K-m values. This isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUTP. We used inhibition patterns, obtained with products to elucidate enzyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequential mechanism with either glucose or ATP as the obligatory first substrates. The ADP inhibition was of mixed type with both ATP and glucose as substrates. (C) 1997 Elsevier B.V.

Influence of membrane-solution interface on the selectivity of SnO2 ultrafiltration membranes

SnO2 supported membranes, presenting 3.0 nm average pore size, have been produced by sol casting on alumina tubular substrate using aqueous colloidal suspensions prepared by sol-gel route. The selectivity and flux throughout SnO2 membrane were analyzed by permeation experiments, using a laboratory tangential filtration pilot equipped with a monotubular membrane. To evaluate the effect of the surface charge at the membrane-solution interface, aqueous salt solutions (NaCl, Na2SO4, CaCl, and CaSO4) of different ionic strength have been filtered and the results correlated with the values of zeta potential measured at several pH. The results show that the retention coefficient is dependent on the electrolyte present in aqueous solution decreasing as: (dication, monoanion) > (monocation, monoanion) approximate to (monocation, dianion) > (dication, dianion). The surface charge and the cation adsorption capacity play a determinant role in these selectivity sequences. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Functional and structural analysis of two fibrinogen-activating enzymes isolated from the venoms of Crotalus durissus terrificus and Crotalus durissus collilineatus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Extracellular tannase from Emericella nidulans showing hypertolerance to temperature and organic solvents

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relação entre as variações da impedância elétrica renal com alterações da taxa de filtração glomerular em cães

Records of the electrical impedance were obtained by means of surface electrodes placed on ventral and dorsal sides of the left kidney of anesthetized dogs. Changes of the renal electrical impedance resulted from alterations of the glomerular filtration rate caused by decrease of blood renal pressure to 80 and 50 mm Hg either due to constriction of the aorta or bleeding. The relation was established also by using physiological infusion. The results showed that changes of the means of renal electrical impedance were in opposite direction to changes in glomerular filtration rate. The electrodes employed were constituted of two parts: one fixed and another adjustable and flexible, allowing good contact with the renal surface independent of the kidney size.

O papel dos rins no equilíbrio ácido-básico em cães submetidos aos efeitos do halotano e hipercapnia

In eighteen dogs, the effects of halothane (0,75% and 1,5%) associated with a normo and hypercapnia (PaCO2 from 30 to 80 mmHg) on acid-base balance were studied. Determinations of creatine clearance, urinary flow, urinary acid excretion, and urinary ammonium excretion were made. Based on the results, it is concluded that halothane associated with hypercapnia decreases the glomerular filtration rate, the urinary flow, the urinary pH and the urinary bicarbonate and sodium excretion, increases the plasmatic bicarbonate concentration, the bicarbonate reabsorbed, the urinary acid excretion and the urinary ammonium excretion, but does not alter the base excess.

Protótipos de filtros para água poluida usada para irrigação de hortaliças e frutiferas

Prototypes A (bidin + broken stones + gravel stones + gross sand + activated coal) and B (bidin + broken stones + gravel stones + gross sand) under hydraulic charge of 35 cm presented outlets that, when extrapolated for larger filters, are sufficient to supply small and medium irrigation projects for vegetables and fruits. (bidin = Rhodia polyester for drainage). Under a charge of 35 cm, filtrate of both prototypes presented a reduction of fecal coliforms, being this reduction higher for A prototypes, but, even in this case, the coliform quantities were superior to the legal limit. Under a decrease of the biochemical oxygen demand an increase of the available oxygen rate; pH and hardness did not present significant changes.

Avaliação tardia da função renal em doentes com paracoccidioidomicose tratados com anfotericina B por meio da determinação do ritmo de filtração glomerular com EDTA Cr51

A late survey of the renal function was performed in eighteen patients with paracoccidioidomycosis treated with amphotericin B, according to the glomerular filtration rate (RFG). The method was compartment analysis by single injection using EDTA Cr51, determined by its 'half biological life' and dosages of blood urea nitrogen and creatinine. The patients were seventeen males and one female. They were from 22 to 76 years old. Ten of these patients received 2 g of amphotericin B and eight of them received 4 g. There were no expressive difference between the two groups, taking into account age, dose in mg/kg of weight/day, time of conclusion of the treatment, urea, creatinine, glomerular filtration was smaller than the normal, and average of the half biological life of the EDTA Cr51 was large than the normal. The achieved results permitted us to consider that the amphotericin B determines deficit of renal function. However, by the present study, it hasn't been possible to affirm if the modifications are definitive.

EFEITOS DA INFUSAO CONTINUA DO PROPOFOL SOBRE A FUNCAO RENAL DO CAO. ESTUDO COMPARATIVO COM O PENTOBARBITAL SODICO

Little research has been done with propofol in relation to renal function. The aim of this study was to evaluate the effects of the continuous infusion of propofol on renal function in dogs. Sixteen dogs, previously anesthetized with pentobarbital sodium (30 mg.kg-1) for surgical preparation, catheterism and monitoring, were studied. The dogs were mechanically ventilated with air and received alcuronium (0.2 mg.kg-1 in bolus and 0.06 mg.kg-1 - maintenance). The following parameters were studied: heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), aortic blood flow (A(o)BF - by electromagnetic flowmeter installed in the ascending aortic), aortic vascular resistance index (A(o)VRI), renal plasma flow (ERPF - by para-aminohipurate clearance), glomerular filtration rate (GFR - by creatinine clearance), effective renal blood flow (ERBF = ERPF/1 - hematocrit), urinary volume (UV), renal vascular resistance (RVR = MAP.80/ERBF.10-3), urinary sodium excretion (UE(Na)), fractionated sodium excretion (FE(Na)), osmolar clearance (C(osm)) and free water clearance (C(H2O)). These parameters were studied at 15 (M1), 30 (M2), 45 (M3) and 60 (M4) min after beginning pentobarbital sodium infusion (5 mg.kg-1.h-1). The dogs were allocated into two groups of eight animals each: G1 (control-pentobarbital sodium) and G2 (propofol). In G1, pentobarbital was given at the four times studied. G2 dogs received the same treatment as G1 dogs at M1 and M2; infusion of pentobarbital was substituted by propofol (3 mg.kg-1 bolus, followed by 12 mg.kg-1.h-1 continuous infusion) at M3 and M4. Profile Analysis was used to analyze the results statistically. In G1 (pentobarbital), there was a significant increase in RVR (M1 < M4) and a decrease in ERPF and ERBF (M1 > M4). In G2 (propofol) there was only a significant increase in A(o)BF (M1 < M2 = M3). In comparison among groups, these was a significant alteration of FE(Na) at M3 (pentobarbital > propofol). It was observed that the continuous infusion of propofol in dogs, at the given doses, did not alter the basic variables of renal function and hemodynamics studied. We concluded that propofol can be one of the drugs of choice to provide base anesthesia in studies of renal function in dogs.

Renal excretion of zinc in normal individuals during zinc tolerance test and glucose tolerance test

The urinary excretion, renal clearance, and tubular reabsorption of zinc were investigated in 30 adult healthy subjects under basal conditions and during the zinc and glucose tolerance tests. After a 12h overnight fast, each subject was submitted to renal clearance of zinc. The procedures were performed between 8.00 and 12.00 a.m., after emptying the bladder and ingestion of 4 ml deionized water/kg body weight at 8.00 a.m. The first urine sample was collected at 10.00 a.m., and the second at 12.00 a.m. A dose of 110 mg ZnSO47H2O was administered orally to each subject, diluted in 20 ml deionized water, at time 0 min. Blood samples were collected from an antecubital vein at times -30, 0, and 30 min and at 30 min intervals up to 240 min. Glucose was administered intravenously (0.5 ml 50%/kg body weight) during the first 3 min of the test, and blood samples were collected from an unconstricted, contralateral, antecubital vein at times -30, 0, 3, 5, 10, 20, 30, 45, 60, and 90 min. The results showed that urinary zinc excretion, and renal zinc clearance were significantly higher during the zinc and glucose tolerance tests than in the control condition. On the other hand, renal zinc clearance was more elevated during tile glucose tolerance test than during the zinc tolerance test. Variations in zinc tubular reabsorption and glomerular filtration rate were not detected. The results suggest that urinary excretion and renal clearance of zinc in healthy subjects increase during acute zinc ingestion and glucose infusion, Although zinc ingestion raised urinary zinc excretion, glucose infusion was more effective in increasing renal zinc clearance. These normal parameters are important in the investigation of diabetic patients with serum and urine zinc changes.

EFEITOS DO SULFATE DE MAGNESIO NA HEMODINAMICA E FUNCAO RENAL DE CAES ANESTESIADOS CORN PENTOBARBITAL SODICO

Background and objectives - The use of magnesium sulphate for the prevention of seizures in pre-eclampsia may induce hypermagnesemia. Clinical and experimental studies are not in agreement about the effects of magnesium on the renal hemodynamics and function. We therefore studied the effects of hypermagnesemia on the renal hemodynamics and function of dogs anesthetized with pentobarbitone. Methods - Sixteen mongrel dogs were anesthetized with pentobarbitone 30 mg.kg-1 and submitted to extracellular ) and mechanical ventilation with room air. The dogs were volume expansion with Ringer's solution (0.4 ml.kg.min allocated into two groups of 8 animals, for the study of renal hemodynamics and function following the administration of 5 mg.kg-1 of pentobarbitone (Group 1 - control or of pentobarbitone associated with magnesium sulphate in the dose (Group 2). The parameters studied were: PAH of 140 mg.kg, administered in 15 minutes, followed by 80 mg.kg-1.h-1 clearance, creatinine clearance, osmolar clearance, free water clearance, renal blood flow, renal vascular resistance, filtration fraction, urinary volume, plasmatic and urinary osmolarity, urinary and fractionary excretion of sodium and potassium, measured at five moments: 15 (M1), 30 (M2), 60 (M3) and 75 (M4) minutes after the first supplementary dose of pentobarbitone and 15 minutes (M5) after the second supplementary dose in Group 1. In Group 2, the moments M3, M4, M5 were 15, 30 and 60 minutes after the priming dose of magnesium sulphate and during the maintenance dose. Results - In Group I no significant changes were observed in renal hemodynamic parameters and creatinine clearance. The extracellular volume expansion increased urinary volume and decreased urinary osmolarity as a consequence of sodium, potassium and free water clearance. The fractionary excretion of sodium was maintained. The plasmatic osmolarity increased. In Group 2, renal hemodynamic parameters and creatinine clearance were also maintained. There was an increase in renal sodium clearance, as detected by the increase in the fractionary excretion of sodium. Conclusions - Magnesium sulphate did not produce significant changes in renal hemodynamics and facilitated the renal excretion of sodium in dogs anesthetized with pentobarbitone.

Produção de biomassa por fungos Filamentosos em meio de vinhaça de cana-de-açúcar suplementado com melaço

Three species of filamentous fungi, Aspergillus niger, Penicillium fellutanum and Mucor hiemalis, were selected and cultivated in vinasse media with different addition of molasses, pasteurized to 85°C for 30 minutes and with pH = 5.0. The microorganisms, previously adapted to the respective medium for 48 hours, from a solution of 107 spores.ml-1, were cultivated in pure and mixed cultures in Erlenmeyer vessel of 500ml, to 30°C, with constant agitation of 170 rpm, for 24, 48 and 72 hours, with four repetition for each samples. The biomass was separated by vacuum filtration in filter Whatman #1 and dried in oven at 105°C until right weight, the obtained liquid was submited to COD analysis. The datas were statistically analysed using a response surface methodology, to improve the effect on the molasses proportion and culture time, in the biomass production by microorganism in research. According to the obtained results (5.02% of molasses, 55.59h, 70% of spores solution of A. niger and 30% of spores solution of P. fellutanum), cultivating was carried out in Microferm Fermentor New Brunswick for 48 hours at 300 rpm, aired at 1v/v/m, using 5 liters of medium added with 5.0% of molasses on the conditions above described. The average of the results obtained (6.81g.l-1) was higher than the confidence interval (5.937 ; 6.369) and was inside the prediction interval (4.471 ; 7.834) both of them significant at 95% by the statistical test employed.

Polybitoxins: A group of phospholipases A2 from the venom of the neotropical social wasp paulistinha (Polybia paulista)

The neotropical wasp Polybia paulista is very aggressive and endemic in south-east Brazil, where it frequently causes stinging accidents. By using gel filtration on Sephadex G-200, followed by ion-exchange chromatography on DEAE-Cellulose under a pH gradient, a group of four toxins (designated as polybitoxins-I, II, lII and IV) presenting phospholipase A2 (PLA2) activities was purified. These toxins are dimeric with mol. wts ranging from 115,000 to 132,000 and formed by different subunits. The four toxins contain very high sugar contents attached to their molecules (22-43% w/w) and presented different values of pH optimum from 7.8 to 9.0; when dissociated, only residual catalytic activities were maintained. The catalytic activities of polybitoxins (from 18 to 771 μmoles/mg per minute) are lower than that of PLA2 from Apis mellifera venom and hornetin from Vespa basalis. The polybitoxins presented a non-linear steady-state kinetic behavior for the hydrolysis of phosphatidylcholine at pH 7.9, compatible with the negative co- operativity phenomena. All of the polybitoxins were very potent direct hemolysins, especially the polybitoxins-III and IV, which are as potent as the lethal toxin from V. basalis and hornetin from Vespa flavitarsus, respectively; polybitoxin-IV presented hemolytic action 20 times higher than that of PLA2 from A. mellifera, 17 times higher than that of neutral PLA2 from Naja nigricolis and about 37 times higher than that of cardiotoxin from Naja naja atra venom.